Keratins are the most abundant proteins in epithelial cells, in which they occur as a cytoplasmic network of 10- 12 nm wide intermediate filaments (IFs). They are encoded by an evolutionarily conserved multigene family, with 54 individual members subdivided into two major types (I and II). The pairwise regulation of type I and II keratin genes reflects a strict heteropolymerization requirement. In addition, keratin genes are regulated in an epithelial tissue-type and differentiation-specific fashion, the functional basis of which is only partly understood. A major role fulfilled by keratin IFs is to act as a resilient scaffold that endows epithelial cells with the ability to sustain mechanical and non-mechanical stresses. Accordingly, inherited mutations affecting the coding sequence of keratins account for a large number of epithelial fragility disorders. Several additional functions, non-mechanical in nature and manifested in a keratin- and context-dependent fashion, have been identified by us and others in recent years. Here we seek to further our understanding of the mechanisms underlying the structural support function of keratins, by focusing largely on K5-K14 filaments, which occur in basal keratinocytes of epidermis and other stratified epithelia. We postulate that the organization of keratin IFs into a cross-linked network, which is essential for function, is determined by intrinsic (i.e., capacity to self-organize) and extrinsic determinants (i.e., involving an interaction with associated proteins). Aim 1 focuses on the contribution of the intrinsic pathway to keratin filament function. In Aim 1a, we will pursue our characterization of the biochemical basis for the remarkable ability of K5-K14 filaments to undergo self-induced bundling. In Aim 1b, we will test the hypothesis that the intrinsic pathway of K5-K14 filament bundling is essential to their structural support role in vivo. To do so we will determine whether mutant forms of K5 proteins can rescue the massive and lethal skin blistering phenotype occurring in K5 null mice. The K5 mutants selected for this purpose are competent for 10 nm filament assembly, but impaired in their ability to form bundles via the intrinsic pathway. Aim 2 focuses on the extrinsic determinants of keratin filament organization in vivo. In Aim 2a, we will identify novel keratin-binding proteins in keratinocytes, and define their impact on keratin IF organization. In Aim 2b, we will define the role of site-specific phosphorylation in regulating the structure, organization, and function of K5-K14 filaments in skin keratinocytes. The proposed research will not only deepen our understanding of the mechanistic underpinnings of the structural support function of keratins under normal and disease conditions, but also help devise new approaches for the treatment of keratin-based diseases. PUBLIC HEALTH RELEVANCE. Keratins are the major cytoskeletal proteins in epithelial cells, in which they provide key structural support. Accordingly, genetic defects in keratin proteins account for a large number of epithelial fragility conditions. This project aims to further our understanding of the structural support role of keratins in skin tissue, with the goal of developing novel approaches for the therapy of keratin-based diseases.